Minimum Allele Frequency Research Articles

Estimating effective population size using RADseq: Effects of SNP selection and sample size.

Effective population size (Ne) is a key parameter of population genetics. However, N e remains challenging to estimate for natural populations as several factors are likely to bias estimates. These factors include sampling design, sequencing method, and data filtering. One issue inherent to the restriction site‐associated DNA sequencing (RADseq) protocol is missing data and SNP selection criteria (e.g., minimum minor allele frequency, number of SNPs). To evaluate the potential impact of SNP selection criteria on Ne estimates (Linkage Disequilibrium method) we used RADseq data for a nonmodel species, the thornback ray. In this data set, the inbreeding coefficient F IS was positively correlated with the amount of missing data, implying data were missing nonrandomly. The precision of Neestimates decreased with the number of SNPs. Mean Ne estimates (averaged across 50 random data sets with2000 SNPs) ranged between 237 and 1784. Increasing the percentage of missing data from 25% to 50% increased Ne estimates between 82% and 120%, while increasing the minor allele frequency (MAF) threshold from 0.01 to 0.1 decreased estimates between 71% and 75%. Considering these effects is important when interpreting RADseq data‐derived estimates of effective population size in empirical studies.

Association of ICOS and CD28 single nucleotide polymorphisms with pulmonary tuberculosis susceptibility

Objective: To investigate the association of inducible co-stimulator (ICOS) and CD28 gene polymorphisms with pulmonary tuberculosis susceptibility. Methods: In this case-control study, from Mar 2015 to Sep 2016, peripheral venous blood of 100 pulmonary tuberculosis patients (pulmonary tuberculosis group) in the Jintan People's Hospital of Changzhou and 100 community physical examination volunteers (health control group) were collected. A total of 56 single nucleotide polymorphisms (SNP) in ICOS and CD28 sequences were selected and SNP genotype and allele frequency were analyzed using the next-generation sequencing technology. Association of these SNP with pulmonary tuberculosis susceptibility was investigated using linkage disequilibrium (LD) analysis and genetic models. Results: Among these 56 SNP, 23 SNP with Hardy-Weinberg equilibrium P (HWE-P) value<0.001 or minimum allele frequency<0.05 were kicked out. The frequencies of T allele and TT genotype of ICOS gene SNP locus (rs55663036), and GG genotype of CD28 gene locus (rs45620941) in tuberculosis group were significantly higher than those in healthy control group (all P<0.05). There was a strong linkage imbalance between rs45620941 at CD28 locus and rs56262258 (r(2)=0.757). Conclusion: The polymorphisms of rs55663036 of ICOS gene and rs45620941 of CD28 gene are significantly associated with the risk of pulmonary tuberculosis.

Impact of the variant allele frequency of ASXL1, DNMT3A, JAK2, TET2, TP53, and NPM1 on the outcomes of patients with newly diagnosed acute myeloid leukemia.

The impact of the allelic burden of ASXL1, DNMT3A, JAK2, TET2, and TP53 mutations on survival remains unclear in patients with newly diagnosed acute myeloid leukemia (AML). The authors assessed bone marrow aspirates from 421 patients with newly diagnosed AML using next-generation sequencing for ASXL1, DNMT3A, JAK2, TET2, and TP53 mutations, defined as the presence of mutations in ASXL1, DNMT3A, JAK2, TET2, or TP53 with a minimum variant allele frequency (VAF) of 5%. A total of 71 patients (17%) had ASXL1 mutations, 104 patients (25%) had DNMT3A mutations, 16 patients (4%) had JAK2 mutations, 82 patients (20%) had TET2 mutations, and 86 patients (20%) had TP53 mutations. Among patients with each mutation, the median VAF of ASXL1 was 34.31% (range, 1.17%-58.62%), the median VAF of DNMT3A was 41.76% (range, 1.02%-91.66%), the median VAF of JAK2 was 46.70% (range, 10.4%-71.7%), the median VAF of TET2 was 42.78% (range, 2.26%-95.32%), and the median VAF of TP53 was 45.47% (range, 1.15%-93.74%). The composite complete response rate was 60%, and was 77% in patients with AML with and without ASXL1, DNMT3A, JAK2, TET2, or TP53 mutations, respectively (P=.006); the median overall survival was 11months and 27months, respectively (P<.001). Multivariate analysis identified age; an antecedent history of dysplasia; white blood cell count; adverse cytogenetic risk; previous treatment with an FLT3 inhibitor; and the VAF of ASXL1, DNMT3A, JAK2, TET2, TP53, and NPM1 mutations by next-generation sequencing as prognostic factors for overall survival. The VAF of ASXL1, DNMT3A, JAK2, TET2, TP53, and NPM1 mutations is associated with worse prognosis in patients with newly diagnosed AML.

Polymorphisms in Inflammatory Genes Modulate the Occurrence of Clinical Complications in Patients with Sickle Cell Disease

Background: Patients (pts) with sickle cell disease (SCD) show a high phenotype variability that is not fully understood. Because inflammation plays a major role in SCD pathophysiology, we hypothesized that single nucleotide polymorphisms (SNPs) in genes of innate and adaptative inflammatory response modulate the occurrence of SCD complications. Objective: to establish associations between SNPs and clinical complications in pts with SCD. Methods: Case-control retrospective study; 500 pts were included, followed at Senegal (n=56), Brazil (n=230) and France (n=214). We analyzed the effect of 20 SNPs in 6 clinical complications: acute chest syndrome (ACS), stroke, leg ulcers, cholelithiasis, osteonecrosis and retinopathy. Using TaqMan 5'-nuclease assay, we genotyped SNPs in genes encoding Toll-like receptor (TLR) 1 (rs4833095), 2 (rs4308099, rs4308100, rs4696480), 6 (rs5743810), 10 (rs11466653, rs11096957), natural killer (NK) group 2 member D (NKG2D) receptor (rs1982536, rs2617160, rs2617169, rs2617170, rs2617171, rs1049174, rs2246809, rs2255336), human leukocyte antigen (HLA)-G (rs9380142), HLA-E (rs2517523), major histocompatibility complex class I polypeptide-related sequence A (MICA, rs1051792) and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) (rs5742909, rs231775). All SNPs had a call rate &gt;90% and minimum allele frequency &gt;1%. We performed analyses of correspondence for indicating associations between SNPs and number of clinical complications. Logistic regressions were used to identify associations between SNPs and each complication, using geographical origin, SCD genotype, rate of transfusions and gender for modeling adjustment; the significance was adjusted for multiple testing using false discovery rate. Comparisons of genotype frequencies with the population of African descent from 1000 genomes database were done by chi-square. Results: Pts were originally from Brazil (n=228), Sub-Saharan Africa (n=200), French West Indies (n=53), North Africa (n=7) and 12 unknown. SCD genotype (available n=498) was SS (n=402), SC (n=46), SB (n=42), SD or SE (n=4). 280 pts were female; median age was 32 years (range 0-69); 184 pts received at least 20 transfusions. 71 pts presented stroke, 200 had at least 1 episode of ACS, 69 had leg ulcers, 271 had cholelithiasis, 150 had retinopathy and 90 osteonecrosis. 97 pts did not present complications, 135 had 1 type of clinical complication, 134 had 2, 94 had 3, 34 had 4 and 6 had 5. 21 pts underwent hematopoietic stem cell transplantation. 11 pts died during follow-up, mostly from ACS and hemorrhagic stroke. Indication of association with number of complications: TLR2 rs4696480 TA, TLR2rs3804099 CC and HLA-Grs9380142 AA were associated with occurrence of 0-1 clinical complications, MICA rs1051792 AA/AG with up to 2 complications and NKG2D rs2617169 AA with 5 complications. Association between genotypes/haplotypes and each complication: no association was found between SNPs and stroke, ACS, leg ulcers and osteonecrosis. Rs9380142 was the only SNP significantly associated with cholelithiasis in the logistic regression additive model. The G allele increased the risk of cholelithiasis (AG x AA, OR 1.57, 95%CI 1.16-2.15; GGxAA, OR 2.47, 95%CI 1.34-4.64; P=0.02). For retinopathy, in the logistic regression additive model, the presence of the A allele decreased the risk of retinopathy for rs2246809 in pts of same origin (AAxGG: OR 0.22, 95%CI 0.09-0.50; AGxGG: OR 0.47, 95%CI 0.31-0.71; P=0.004), rs2617160 (ATxTT: OR 0.67, 95%CI 0.48-0.92; AAxTT: OR 0.45, 95%CI 0.23-0.84; P=0.04) and rs2617169 in pts of same SCD genotype (AAxTT: OR 0.33,95%CI 0.13-0.82; ATxTT: OR 0.58, 95%CI 0.36-0.91, P=0.049). No haplotype was associated with complications. The genotype distribution of SNPs rs4696480, rs3804099, rs1051792 and rs2617169 differed significantly from the population of African descent from the 1000 genomes database. Discussion: We have previously shown that TLR2 rs4696480 TA decreases occurrence of bacterial infections in SCD; in this study, TA is also associated with less complications. Also, HLA-G rs9380142 AA had less complications and cholelithiasis, and 3 SNPs in NKG2D modulated occurrence of retinopathy. TLR and NKG2D may be activated by heat shock proteins, released in ischemia-reperfusion injury that occurs in SCD. Our findings help to better understand the role of inflammation in phenotype heterogeneity in SCD. Disclosures No relevant conflicts of interest to declare.

Shared Genomic Alterations in Patients with Co-Existing Myeloproliferative Neoplasms and Angioimmunoblastic T-Cell Lymphoma

Background: Myeloproliferative neoplasia (MPN) and angioimmunoblastic T-cell lymphoma (AITL) are two distinct hematologic cancers. However, we have shown that patients with both malignancies probably occur with frequencies higher than expected in the background population (1,2). AITL tumors show a high frequency (30-70%) of mutations in RHOA and epigenetic modifier genes such as DNMT3A, TET2 and IDH2. The latter genomic alterations are similar to those found in myeloid diseases such as MPN. This observation has raised questions on whether the two hematologic malignancies may share genomic aberrations (e.g. at progenitor level) suggesting a possible pathogenetic relationship between these diseases. Aim: To further investigate these questions, we explored the mutational landscape in MPN and AITL tumors occurring in the same host. Methods: From a Danish cohort of 97 patients with co-existing MPN and lymphoma identified through the Danish Lymphoma and Pathology Registries and immunohistochemically validated by tertiary center hematopathologists, five patients with MPN and concurrent and/or subsequent AITL were included. Paired DNA samples obtained from lymph node biopsies (AITL component), bone marrow aspirates (MPN component) and saliva specimens (normal reference DNA) were analyzed by whole exome sequencing. A minimum variant allele frequency threshold of 10% was applied. However, mutations in epigenetic modifier genes, RHOA and JAK2, were also included, if found at slightly lower frequencies. Results: Figure 1 shows an overview of the identified mutations. At a VAF% cut-off value of 10 and a median coverage of 30, mutations shared between the MPN and AITL specimens were found within the DNMT3A, TET2, and IDH2 genes. Mutations in the JAK2 gene were identified in all MPN and in some of the AITL samples. NOTCH2 mutations were frequent and some were shared between the MPN and AITL samples. Two patients with essential thrombocytosis and AITL had the same NOTCH2 (C19W) and NCOR1 (K178N) mutation. Most of the identified genomic alterations were inactivating missense mutations. Conclusion: Co-existence of MPN and AITL occurs and may not be a random event. We studied tumor tissues from patients diagnosed with both diseases, and identified recurrent shared and non-shared mutations in samples from both types of cancers. These mutations may contribute to the development of the two malignancies in the same host, partly through common steps at precursor level (e.g. DNMT3A) and partly more downstream at a more lineage-specific level (e.g. IDH2). The functional role of the observed NOTCH2 and NCOR1 mutations is currently under investigation. Holst J et al. Blood 2017, 130 (S1): 1525 Frederiksen H et al. Blood 2011;118(25):6515-6520 Disclosures Tam: Takeda: Consultancy; Paragon Genomics: Consultancy.

Targeted next-generation sequencing of circulating-tumor DNA for tracking minimal residual disease in localized colon cancer.

A high percentage of patients diagnosed with localized colon cancer (CC) will relapse after curative treatment. Although pathological staging currently guides our treatment decisions, there are no biomarkers determining minimal residual disease (MRD) and patients are at risk of being undertreated or even overtreated with chemotherapy in this setting. Circulating-tumor DNA (ctDNA) can to be a useful tool to better detect risk of relapse. One hundred and fifty patients diagnosed with localized CC were prospectively enrolled in our study. Tumor tissue from those patients was sequenced by a custom-targeted next-generation sequencing (NGS) panel to characterize somatic mutations. A minimum variant allele frequency (VAF) of 5% was applied for variant filtering. Orthogonal droplet digital PCR (ddPCR) validation was carried out. We selected known variants with higher VAF to track ctDNA in the plasma samples by ddPCR. NGS found known pathological mutations in 132 (88%) primary tumors. ddPCR showed high concordance with NGS (r = 0.77) for VAF in primary tumors. Detection of ctDNA after surgery and in serial plasma samples during follow-up were associated with poorer disease-free survival (DFS) [hazard ratio (HR), 17.56; log-rank P = 0.0014 and HR, 11.33; log-rank P = 0.0001, respectively]. Tracking at least two variants in plasma increased the ability to identify MRD to 87.5%. ctDNA was the only significantly independent predictor of DFS in multivariable analysis. In patients treated with adjuvant chemotherapy, presence of ctDNA after therapy was associated with early relapse (HR 10.02; log-rank P < 0.0001). Detection of ctDNA at follow-up preceded radiological recurrence with a median lead time of 11.5 months. Plasma postoperative ctDNA detected MRD and identified patients at high risk of relapse in localized CC. Mutation tracking with more than one variant in serial plasma samples improved our accuracy in predicting MRD.

Development of a new 32-plex InDels panel for forensic purpose

Insertion/deletion polymorphisms (InDels) have attracted more and more attention of forensic researchers because of their low mutation rate and small amplicons. In the face of challenging forensic cases and degraded DNA, InDels break through the limitations of traditional STRs and provided a new direction for forensic identification. In this study, a multiplex panel consisting of 32 InDels and amelogenin was established and the InDels were selected with minimum allele frequencies (MAF) ≥ 0.4 in Chinese Han population based on the 1000 Genomes Project and published articles. The 32-plex InDels assay was evaluated using the southern Chinese Han population from Hubei province. All markers were proved to be highly polymorphic with an average observed heterozygosity (Hobs) of 0.4885. The combined power of discrimination (CPD) and the combined probability of paternity exclusion (CPE) were 0.999999999999966 and 0.9982, respectively. Further validation studies indicated that the 32-plex InDels assay was highly sensitive to DNA template amounts as low as 31.25 pg and was effective for degraded DNA. The obtained allele frequencies from this study were compared with published data of other populations.

Assessing Genetic Diversity for a Pre-Breeding Program in Piaractus mesopotamicus by SNPs and SSRs.

The pacu (Piaractus mesopotamicus) is a Neotropical fish with remarkable productive performance for aquaculture. Knowledge of genetic resources in Neotropical fish is essential for their applications in breeding programs. The aim of this study was to characterize the genetic diversity of seven farmed populations of pacu which will constitute the basis for a broodstock foundation for coming breeding programs in Brazil. Analysis of one wild population (Paraná River) was used as a reference to compare genetic parameters in the farmed populations. The analyses were performed using 32 single-nucleotide polymorphisms (SNP) and 8 simple sequence repeat (SSR) markers. No significant differences in genetic diversity between populations estimated through the number of alleles and allelic richness, observed heterozygosity, expected heterozygosity, and minimum allele frequency were detected (p > 0.05). Low genetic diversity was observed in all farmed stocks and the wild population. Moreover, we detected low genetic structure when comparing farmed and wild populations for SNPs (FST = 0.07; K = 3) and SSRs (FST = 0.08; K = 2). Analysis of molecular variance (AMOVA) demonstrated that genetic variation was mostly within populations. Kinship analysis showed that most fish farms included related individuals at a proportion of at least 25%. Our results suggest that the basal broodstock for pacu breeding programs should be founded with individuals from different fish farms for higher genetic diversity and to avoid inbreeding risks.

Association between zinc finger protein 350 gene polymorphism and early onset breast cancer among women from different ethnic groups in Xinjiang

Objective To detect the variants of zinc finger protein 350 (ZNF350) gene in exon of Xinjiang women with early onset breast cancer, and investigate the relationship between Polymorphism and the early onset breast cancer of women. Methods Exon region of ZNF350 gene are detected via direct sequencing in Xinjiang minority and Han nationality women with early onset breast cancer (age: 30-40 years old, separately 80 cases), The impact of Non-synonymous variants on protein function are predicted by using Polyphen and SIFT software, and then analyzing the relationship between Polymorphism genotypes and clinical pathological features. Results (1) The test results of ZNF350 gene exon region mutation: Nine mutations are detected (referring to database minimum allele frequency MAF>1% before the trial), including 5 missense mutations and 4 synonymous mutations, which located in EXON3, EXON4 and EXON5. Among them, the Uighur nationality are detected eight locus mutations, and the han nationality have seven locus mutations, which including 4 missense mutations respectively. (2) Using Polyphen and SIFT software to predict protein function: The result show that the common mutation sites rs4987241 and rs2278415 of minority and Han nationality patients with early-onset breast cancer are deleterious mutations, which may affect the ZNF350 protein function. (3) The relevance between distribution of polymorphic genotype and clinical pathological features: ① There is a certain correlation between rs4986773 and axillary lymph node status of breast cancer in minority (r=0.234, P 0.05). Conclusion Among early-onset breast cancer in Xinjiang women, there is a certain correlation between ZNF350 gene polymorphism and menarche age, tumor size, axillary lymph node status and triple-negative breast cancer, which can be used as one of the predictors to guide the early-onset breast cancer individualize treatment in Xinjiang women. Key words: Early-onset breast cancer; Zinc finger protein 350 gene; Single nucleotide polymorphism

Analysis of single-nucleotide polymorphism of Sonic hedgehog signaling pathway in non-syndromic cleft lip and/or palate in the Chinese population

To study the relationship between Sonic hedgehog (Shh) associated single-nucleotide polymorphism (SNP) and non-syndromic cleft lip and/or palate (NSCL/P), and to explore the risk factors of cleft lip and/or palate. Many studies suggest that the pathogenesis of NSCL/P could be related to genes that control early development, in which the Shh signaling pathway plays an important role. Peripheral blood was collected from 197 individuals (100 patients with NSCL/P and 97 healthy controls). Haploview software was used for haplotype analysis and Tag SNP were selected, based on the population data of Han Chinese in Beijing of the international human genome haplotype mapping project. A total of 27 SNP were selected for the 4 candidate genes of SHH, PTCH1, SMO and GLI2 in the Shh signaling pathway. The genotypes of 27 SNP were detected and analyzed by Sequenom mass spectrometry. The data were analyzed by chi-squared test and an unconditional Logistic regression model. The selected SNP basically covered the potential functional SNP of the target genes, and its minimum allele frequency (MAF) was >0.05: GLI2 73.5%, PTCH1 91.0%, SMO 100.0%, and SHH 75.0%. It was found that the genotype frequency of SNP (rs12674259) located in SMO gene and SNP (rs2066836) located in PTCH1 gene were significantly different between the NSCL/P group and the control group. Linkage disequilibrium was also found on 3 chromosomes (chromosomes 2, 7 and 9) where the 4 candidate genes were located. However, in the analysis of linkage imbalance haplotype, there was no significant difference between the disease group and the control group. In China, NSCL/P is the most common congenital disease in orofacial region. However, as it is a multigenic disease and could be affected by multiple factors, such as the external environment, the etiology of NSCL/P has not been clearly defined. This study indicates that Shh signaling pathway is involved in the occurrence of NSCL/P, and some special SNP of key genes in this pathway are related to cleft lip and/or palate, which provides a new direction for the etiology research of NSCL/P and may provide help for the early screening and risk prediction of NSCL/P.

IL7RA rs6897932 Polymorphism is Associated with Better CD4+ T-Cell Recovery in HIV Infected Patients Starting Combination Antiretroviral Therapy.

Interleukin-7 receptor subunit alpha (IL7RA) rs6897932 polymorphism IS related to CD4+ recovery after combination antiretroviral therapy (cART), but no studies so far have analyzed its potential impact in patients with very low CD4+ T-cells count. We aimed to analyze the association between IL7RA rs6897932 polymorphism and CD4+ T-cells count restoration in HIV-infected patients starting combination antiretroviral therapy (cART) with CD4+ T-cells count <200 cells/mm3. We performed a retrospective study in 411 patients followed for 24 months with a DNA sample available for genotyping. The change in CD4+ T-cells count during the follow-up was considered as the primary outcome. The rs6897932 polymorphism had a minimum allele frequency (MAF) >20% and was in Hardy–Weinberg equilibrium (p = 0.550). Of 411 patients, 256 carried the CC genotype, while 155 had the CT/TT genotype. The CT/TT genotype was associated with a higher slope of CD4+ T-cells recovery (arithmetic mean ratio; AMR = 1.16; p = 0.016), higher CD4+ T-cells increase (AMR = 1.19; p = 0.004), and higher CD4+ T-cells count at the end of follow-up (AMR = 1.13; p = 0.006). Besides, rs6897932 CT/TT was related to a higher odds of having a value of CD4+ T-cells at the end of follow-up ≥500 CD4+ cells/mm3 (OR = 2.44; p = 0.006). After multiple testing correction (Benjamini–Hochberg), only the increase of ≥ 400 CD4+ cells/mm3 lost statistical significance (p = 0.052). IL7RA rs6897932 CT/TT genotype was related to a better CD4+ T-cells recovery and it could be used to improve the management of HIV-infected patients starting cART with CD4+ T-cells count <200 cells/mm3.

Influence of single nucleotide polymorphism of Wilms tumor 1 gene on the efficacy and clinical prognosis of elderly patients with acute myeloid leukemia

Objective To evaluate the influence of single nucleotide polymorphism of Wilms tumor 1(WT1)gene rs16754 on the chemosensitivity and clinical outcomes of elderly patients with acute myeloid leukemia(AML). Methods A total of 178 AML patients aged 60 years and over who received cytarabine-based chemotherapy were enrolled in this retrospective study.The peripheral blood was extracted from 178 AML patients receiving chemotherapy for DNA preparation and study.And bone marrow specimens were collected in 65 AML patients before chemotherapy.The Wilms' Tumor-1(WT1)rs16754 polymorphism was detected by PCR-RFLP method.The association between genotypes and other variables were analyzed by using Logistic regression model.Variables were adjusted by Cox regression analysis. Results The locus of WT1 gene rs16754 is located in coding region of WT1 gene.The genotype frequency and distribution of the studied population were 55.62%(99/178)in GG, 37.64%(67/178)in GA, and 6.74%(12/178)in AA, with minimum allele frequencies of 0.26.The distributions of the three genotypes were in accordance with Hardy-Weinberg Equilibrium(P=0.884). There was no statistical difference in the data distribution of the genotypes on clinical indexes at baseline.Overall survival time(OS)was longer in patients with allele A and genotype GA plus AA[2.73 years(95%CI: 1.03-5.11 years)]than in patients with GG genotype[1.64 years(95%CI: 0.71-4.34), (P=0.003)]. The replase free survival(RFS)was longer in patients with allele A and genotype GA plus AA[2.06 years(95%CI: 0.95-4.87)]than in patients with GG genotype[1.12 years(95%CI: 0.56-4.11), P=0.032)]. Adjusted by using multivariate Cox regression analysis, GA plus AA genotypes still showed a better effect on OS(HR=0.51, P=0.013)than did GG genotypes.In the 65 pretreatment bone marrow specimens, the expression level of WT1 mRNA in bone marrow cells was higher in patients with GG genotype than in patients with GA plus AA genotype(P<0.001). Conclusions Among elderly AML patients treated by cytarabine-based chemotherapy, the WT1 rs16754 may impact the clinical prognosis of AML patients by influencing the mRNA expression of WT1. Key words: Leukemia, myeloid; Polymorphism, single nucleotide; Treatment outcome

The first genetic linkage map for Fraxinus pennsylvanica and syntenic relationships with four related species.

The genetic linkage map for green ash (Fraxinus pennsylvanica) contains 1201 DNA markers in 23 linkage groups spanning 2008.87cM. The green ash map shows stronger synteny with coffee than tomato. Green ash (Fraxinus pennsylvanica) is an outcrossing, diploid (2n = 46) hardwood tree species, native to North America. Native ash species in North America are being threatened by the rapid spread of the emerald ash borer (EAB, Agrilus planipennis), an invasive pest from Asia. Green ash, the most widely distributed ash species, is severely affected by EAB infestation, yet few genomic resources for genetic studies and improvement of green ash are available. In this study, a total of 5712 high quality single nucleotide polymorphisms (SNPs) were discovered using a minimum allele frequency of 1% across the entire genome through genotyping-by-sequencing. We also screened hundreds of genomic- and EST-based microsatellite markers (SSRs) from previous de novo assemblies (Staton et al., PLoS ONE 10:e0145031, 2015; Lane et al., BMC Genom 17:702, 2016). A first genetic linkage map of green ash was constructed from 90 individuals in a full-sib family, combining 2719 SNP and 84 SSR segregating markers among the parental maps. The consensus SNP and SSR map contains a total of 1201 markers in 23 linkage groups spanning 2008.87cM, at an average inter-marker distance of 1.67cM with a minimum logarithm of odds of 6 and maximum recombination fraction of 0.40. Comparisons of the organization the green ash map with the genomes of asterid species coffee and tomato, and genomes of the rosid species poplar and peach, showed areas of conserved gene order, with overall synteny strongest with coffee.

A Polymorphism in Toll-like Receptor 2 Gene Is Associated with Occurrence of Bacterial Infections in Sickle Cell Disease Patients

Background: Sickle cell disease (SCD) patients (pts) are at increased risk of infection. This susceptibility is partially explained by the loss of spleen function, but other factors such as inflammatory status might also play a role. Despite adequate immunization and antimicrobial prophylaxis, bacterial infection remains a major cause of morbidity and mortality in SCD pts. Toll-like receptors (TLR), transmembrane proteins that recognize a wide range of pathogen molecular patterns, are key receptors in innate immune response. The association among TLR and infections in SCD has scarcely been explored.Objective: To analyze if functional polymorphisms in TLR genes can modulate the susceptibility to bacterial infections in SCD pts.Methods: Case-control retrospective study; 430 SCD pts followed in university hospitals in Brazil (n=196), France (n=180, mainly from Subsaharan Africa and French West Indies) and Senegal (n=54) were included. Sites of bacterial infection were lower respiratory system, abdominal, upper urinary system, bone/joints, central nervous system, blood stream, or any site for tuberculosis. For pts who underwent hematopoietic stem cell transplant (HSCT), only bacterial infections before HSCT were considered. All pts were tested for 7 single nucleotide polymorphisms (SNP) in TLR-1(rs4833095), TLR-2 (rs4696480, rs3804099, rs3804100), TLR-6 (rs5743810) and TLR-10 (rs11466653, rs11096957) using TaqMan 5'-nuclease assay. Univariate analysis was performed with chi-square or Mann-Whitney for categorical and continuous variables respectively. Associations were measured by odds ratio (OR). For SNP associations, significant P-value corrected by Bonferroni (cP) was 0.007; for genetic models, cP was 0.01.Results: Median age of cohort was 30 years (range: 2-70) and 87% were ≥18 years; 57% of pts were female. SCD genotype was SS in 81%, SC in 11%, SB in 7% and other in 1%. Fifty-eight percent received hydroxyurea and 79% received red blood cell transfusions; seven pts underwent HSCT. Eleven pts died during follow-up, mostly from acute chest syndrome and hemorrhagic stroke. All pts had access to immunization against encapsulated bacteria and to penicillin prophylaxis. One hundred ninety-five pts (45%) did not present any bacterial infection, whereas 235 (55%) pts presented at least one episode of bacterial infection, of which 20% had ≥ 3 episodes. Bacterial infections were mainly respiratory (51%), bone/joints (23%) and urinary (20%). In 106 cases, etiological agents were identified; most common agents were Escherichia coli, Streptococcuspneumoniae, Mycobacterium tuberculosis and Salmonella spp. In univariate analysis, there was no association among occurrence of infections and gender, origin and age distribution. Gene analysis: all tested SNPs had a typing rate >90% and minimum allele frequency >1%. The TLR-2 SNP rs4696480 (n=425) showed significant association with infection (p<0.001). Next, we tested genetic models for this association. Distribution of rs4696480 genotypes was: infected - AA=35%, TA=50%, TT=15%; non infected - AA=28%, TA=67%, TT=5%. In the overdominant model, the heterozygous genotype (TA) occurred significantly more in non infected (67%) than in infected pts (50%) compared with TT+AA (OR 0.5, 95%CI 0.34-0.75, P<0.001). Also, in the recessive model, genotype TT occurred significantly more in infected (15%) than in non infected pts (5%) compared with TA+AA (OR 3.18, 95%CI 1.53-6.61, P<0.001). Furthermore, in comparison with an African/African-American population described in the 1000 genomes database (n=661), TA genotype was more frequent in SCD pts, and TT genotype was significantly less frequent. Other TLR SNPs genotype and haplotype analysis did not show any significant association with occurrence of infections.Discussion: In SCD pts, TLR-2 rs4696480 TA genotype might be protective against bacterial infections, whereas TT genotype might increase risk of such infections. Previous reports demonstrated higher secretion of inflammatory factors in cells from AA individuals, lower occurrence and severity of immune diseases in T carriers. TA genotype might stand between deleterious effects of over inflammatory response (AA genotype) and under response (TT genotype) to infectious agents. DisclosuresNo relevant conflicts of interest to declare.

Whole Exome Sequencing of Residual Disease in Multiple Myeloma: Searching for Novel Therapeutic Targets

Introduction:Multiple myeloma (MM) is a plasma cell dyscrasia causing damage of multiple organs with fatal consequences for patients. Despite the success of modern therapies eliminating a vast bulk of the aberrant cells, surviving residual clones eventually lead to the relapse of the disease. Accumulation of genomic alterations during the stage of minimal residual disease (MRD) likely contributes to a selective grow advantage and survival under the drug pressure. Identification of specific mutations in MM patients with MRD can provide unique opportunities to target the residual plasma cell clones. Here we present the first whole exome sequencing (WES) analysis of 22 MM samples of patients with MRD that identified 814 mutated genes with 4% of genes previously implicated in the pathogenesis of MM.Methods:Aberrant plasma cells (A-PCs) and peripheral blood (PB) were collected from patients after signing informed consent form. Presence of MRD was assessed with EuroFlow protocol and A-PCs were sorted out from bone marrow according to their pathological immunophenotype based on the expression of antigens CD38, CD45, CD19 and CD56. DNA from A-PCs was isolated and amplified by Repli-g Single cell kit (QIAGEN). Sequencing libraries were prepared using SureSelect Human All Exon V6 Kit (Agilent Technologies) and sequenced by Macrogen Inc. on Illumina HiSeq 4000 platform with average coverage 50x and 2x 100bp read length. Sequencing data were processed using the Bcbio framework following the standard workflow for tumor-matched-normal studies. Specifically, reads were mapped to the human reference genome GRCh37, successively marking duplicates using Picard. Germline mutations were identified using GATK HaplotypeCaller, whereas somatic mutations were identified using MuTect2 reporting as significant variants observed in at least 5 reads and minimum allele frequency of 10%. Variants in homopolymer regions longer than 5 nucleotides were filtered out. The final set of calls were further characterised by assessing their functional impact using snpEff and by annotating each variant using data from 1000 Genomes Phase 3, ExAC, and ClinVar. We then used OncodriveCLUST to identify putative oncogenic genes, and later compared these results with a literature curated list of MM driver genes (Weaver & Tariman, 2017).Results:Our dataset comprises 22 samples from 21 patients (one patient was sampled in two time points) with MM MRD, who received bortezomib-based regimen (age 41-71, average 59 years, 11/22 males, 10/22 females). 8 patients reached complete response, 9 patients had very good partial response and 4 patients had partial response. In our analysis, we detected 1,014 tumour somatic variants (8-287 per sample, median 36), most of them being missense mutations (676/1014), splice site mutations (145/1014) and frameshift insertions (134/1014). The variants affected a total of 814 genes, 97 genes were shared in at least two samples. The most frequently mutated genes were KIAA1211 (11/22), the immunoglobulin gene IGLV3-1 (8/22), apoptotic chromatin condensation inducer ACIN1 (7/22) and CCR4-associated factor 3 CNOT3 (7/22). We also identified 32 genes known to be mutated in MM in 64% of our samples (14/22). We found mutations shared by at least 2 samples in KRAS (4/22), DIS3 (3/22), TRAF3 (3/22), NRAS (2/22), ANK2 (2/22), BRAF (2/22) and RBM15 (2/22). Further analysis with OncodriveCLUST identified 18 putative oncogenic genes (FDR < 0.1), including KRAS, DIS3, ACIN1.Conclusion:We presented the first whole exome study of MM MRD, providing a characterisation of the mutations observed in A-PCs. We overcame problem with low amount of A-PCs in this disease stage by using whole genome amplification and a highly customised bioinformatic analysis pipeline. Our study suggests that A-PCs are characterised by new MM MRD specific set of mutated genes, along with the presence of mutations in well-known multiple myeloma cancer driver genes. This offers a great potential for design of novel precise treatments targeting MRD after standard MM therapies.Supported by Ministry of Health of the Czech Republic (17-30089A, CZ-DRO-FNOs/2016) and Ministry of Education of the Czech Republic (SGS18/PřF/2017-2018) DisclosuresKryukov:JSC BIOCAD: Employment. Maisnar:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees. Hajek:Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding; Novartis: Research Funding.

Association of TLR8 and TLR9 polymorphisms with tuberculosis in a Chinese Han population: a case-control study

BackgroundToll-like receptor (TLR) single nucleotide polymorphisms (SNPs) have been associated with regulation of TLR expression and development of active tuberculosis (TB). The objectives of this study were to determine whether TLR8 and TLR9 SNPs were associated with the development of latent TB infection (LTBI) and the subsequent pulmonary TB (PTB) in a Chinese Han population.MethodsTwo independent samples were enrolled. The first sample contained 584 TB cases and 608 controls; the second sample included 204 healthy controls, 201 LTBI subjects and 209 bacteria-confirmed active PTB patients. Three SNPs (rs3764880, rs187084 and rs5743836) were genotyped. The associations between the SNPs and risk of LTBI or PTB were investigated using unconditional logistic regression analysis.ResultsThe A-allele of TLR8 rs3764880 SNP was protective against the development of TB in males (A vs G, OR = 0.58, 95%CI = 0.37–0.91). The AA genotype of rs3764880 SNP was found to increase the risk of PTB among females with an OR of 4.81 (1.11–20.85). The G allele of TLR9 SNP rs187084 was found to increase the risk of PTB (G vs A, P = 0.01, OR = 1.48, 95% CI = 1.10–2.00), the significance was also observed under dominant genetic models. The GA-genotype of TLR9 rs187084 SNP was found to increase the risk of PTB with an OR of 1.68 (1.07–2.65), but was found to decrease the risk of MTB infection with an OR = 0.64 (0.41–0.98). TLR9_rs5743836 SNP was excluded from the data analyses, because the minimum allele frequency was< 1%.ConclusionsOur findings in two independent samples indicated that SNPs in TLR8 and TLR9 were associated with the development of TB, and highlight that SNPs may have different effects on disease pathogenesis and progression.

A9357 The genetic polymorphisms of TWIK-related acid-sensitive K+ channel-3 (TASK-3) were associated with obstructive sleep apnea in patients with hypertension

Objectives: TWIK-related acid-sensitive K+ channel-3 was reported to be a risk factor for OSA in animal models. The present study aimed to evaluate the relationship between human TASK-3 gene polymorphisms and obstructive sleep apnea in patients with hypertension. Methods: 176 subjects with non-OSA and 169 subjects with severe OSA by full night polysomnography were selected. And we chose three represent single nucleotide polymorphisms (SNPs) such as rs2615374, rs3808403 and rs888345, according to principle of linkage disequilibrium (r2 > 0.8) and minimum allele frequency (MAF) > 5%. Blood samples were collected from all subjects and genotyping of the 3 SNPs was performed by Kompetitive Allele Specifc PCR. Results: The significant differences in additive model (P = 0.047), alleles (P = 0.014) and dominant model (P = 0.02) of rs2615374 were observed between severe and non OSA subjects. The distributions of additive (P = 0.005) and dominant model (P = 0.015) of rs3808403 were also significantly different between two groups. CT+TT genotype of rs3808403 was considered as risk factors for Severe OSA (CT+TT: [OR] = 2.09, 95%CI: 1.13–3.88, P = 0.019). In addition, mutant group of rs2615374 and rs3808403 showed significantly higher triglyceride levels than wild group. Conclusion: Our study suggested that the polymorphisms of rs2615374 and rs3808403 in the TASK-3 gene were associated with OSA, and CT+TT genotype of rs3808403 locus was a risk factor for severe OSA among the Chinese people. However, further studies should be conducted to confirm this association.

Evaluating DNA evidence in a genetically complex population

In forensic genetics, the likelihood ratio (LR), measuring the value of DNA profile evidence, is computed from a database of allele frequencies. Here, we address the choice of database and adjustments for population structure and sample size in the context of Brazil. The Brazilian population underwent a complex process of colonization, migration and mating, which created an admixed genetic composition that makes it difficult to obtain an appropriate database for a given case. National databases are now available, as well as databases for many Brazilian states. However, those databases are not statistically random samples, and state boundaries may not accurately reflect the sub-structuring of genetic diversity. We compared the LR calculated using the relevant state-specific database with the statistics calculated when a national database and when international databases were used. We evaluated two methods of adjustment for population structure, due to Wright [13] and Balding and Nichols [14]. We also considered two adjustments for database sample size: the Balding size bias correction [15] and a minimum allele frequency [16]. Our results show that the use of a national database with the Balding and Nichols adjustment and θ = 0.002 generated lower LR values than did the state-specific database in more than 50% of the profiles simulated using the state-based allele frequencies, while θ = 0.01 produced lower LRs for more than 90% of the profiles. We conclude that the utilization of a national database for Brazilian cases can be justified in association with the appropriate adjustment for population structure.

ASSOCIATION OF TASK-3 GENETIC POLYMORPHISMS WITH OBSTRUCTIVE SLEEP APNOEA

Objective: Objectives: To evaluate the relationship between human TASK-3 gene polymorphisms and obstructive sleep apnea (OSA), and to explore the genetic mechanism of OSA Design and method: Methods: 176 subjects with non-OSA and 169 subjects with severe OSA by full night polysomnography were selected. We chose three represent single nucleotide polymorphisms (SNPs) such as rs2615374, rs3808403 and rs888345, according to principle of linkage disequilibrium (r2 > 0.8) and minimum allele frequency (MAF) > 5%. Blood samples were collected from all subjects and genotyping was performed on DNA extracted from blood cells by Kompetitive Allele Specifc PCR. Results: The significant differences in additive model (GG 39.2% vs 52.1%, AG 47.5% vs 39.9%,AA 13.3% vs 8.0%, P = 0.047), alleles (A 37% vs 27.9%,G 63% vs 72.1%, P = 0.014) and dominant model (GG 39.2% vs 52.1%,GA+AA 60.8% vs 47.9%, P = 0.02) of rs2615374 were observed between severe and non OSA subjects. The distributions of additive (CC 23.3% vs 35.9%,CT 61.6% vs 43.1%,TT 15.1% vs 21.0%, P = 0.005) and dominant model (CC 23.3% vs 35.9%,CT+TT 76.7% vs 64.1%, P = 0.015) of rs3808403 were also significantly different between two groups. CT+TT genotype of rs3808403 was considered as risk factors for Severe OSA (CT+TT: [OR] = 2.09, 95%CI: 1.16–3.78, P = 0.014). In addition, Mutant group of rs2615374 and rs3808403 showed significantly higher triglyceride levels than wild group. Conclusions: Conclusions: The polymorphisms of rs2615374 and rs3808403 in the TASK-3 gene were associated with incidence of OSA, and CT+TT genotype of rs3808403 locus was a risk factor for severe OSA among the Chinese people. However, further studies should be conducted to confirm this association.

Transcriptome based SNP discovery and validation for parentage assignment in hatchery progeny of the European abalone Haliotis tuberculata

Selective breeding strategies require pedigree information over generations, but many species produced in aquaculture are too small to be physically tagged at early stages. Consequently, maintaining a sufficient number of separate families is often needed but costly and logistically difficult. Alternatively, parentage assignment can be obtained using DNA markers. We developed a panel of single nucleotide polymorphism (SNP) markers for the European abalone Haliotis tuberculata using an existing transcriptomic resource. An initial set of 2,176,887 SNPs was filtered to select 500 for high throughput genotyping. Of these, 298 SNPs were amplified in at least 90% of our H. tuberculata samples, consisting of a mixed family cohort (945 offspring) generated by crossing 40 abalones, and 5 full-sib training families (70 offspring). Based on amplification success among parents, minimum allele frequency and checks carried out against the training families, a subset of 123 markers was used to carry out parentage assignment in our mixed family cohorts. Maximum likelihood and exclusion-based methods of parentage assignment yielded consistent results, allowing parentage to be assigned in 98.9% of the studied progeny. Optimization of markers suggests that the 60 most informative SNPs may be sufficient for 95% assignment success in these progeny. The panel was also used to estimate effective population size, and revealed a low Ne due to high variance of reproductive success between parents. Our panel could be used to estimate genetic parameters of traits in mixed family cohorts, an essential stage to initiate selective breeding in H. tuberculata. It could also be useful tool in the context of monitoring stock enhancement and population genetics studies.